Method of altering blood sugar levels using non-transformed human pancreatic cells that have been expanded in culture

ABSTRACT

The present invention provides a method for producing an expanded non-transformed cell culture comprising the steps of: (1) preparing partially purified, minced tissue; (2) concentrating the resulting cells and tissue pieces; (3) resuspending the concentrated tissue cells and pieces in a culture medium capable of supporting sustained cell division that is contained in a culture vessel; (4) incubating the cells; and (5) passaging the cells periodically. The present invention further provides clonal strains of cells derived from the above-mentioned cell culture, medium and conditioned medium designed for the culturing of such cells, including pancreatic, thyroid, parathyroid, and parotid cells, and the use of cultured pancreatic cells to form pancreatic pseudotissues composed of matrix-embedded aggregated (pseudoislets) or individual cells, to treat blood sugar disorders in mammals, and to test for cytotoxicity and autoimmune activities with reference to pancreatic endocrine cells.

This is a of copending applications(s) Ser. No. 08/083,772 filed on Jun.30, 1993 now abandoned, which in turn is a continuation-in-part of Ser.No. 08/044,010, filed Apr. 8, 1993, now abandoned.

FIELD OF THE INVENTION

This invention relates to a method and medium for the culturing ofdifferentiated mammalian cells.

BACKGROUND OF THE INVENTION

Many kinds of cells can be grown in culture, provided that suitablenutrients and other conditions for growth are supplied. Thus, since 1907when Harrison noticed that nerve tissue explanted from frog embryos intodishes under clotted frog lymph developed axonal processes, scientistshave made copious use of cultured tissues and cells from a variety ofsources. Such cultures have been used to study genetic, physiological,and other phenomena, as well as to manufacture certain macromoleculesusing various fermentation techniques known in the art. In studies ofmammalian cell biology, cell cultures derived from lymph nodes, muscle,connective tissue, kidney, dermis and other tissue sources have beenused. Generally speaking, the tissue sources that have been mostsusceptible to the preparation of cell cultures for studies arederivatives of the ancestor mesodermal cells of early development.Tissues that are the progeny of the ancestor endodermal and ectodermalcells have only in recent years become amenable to cell culture, of alimited sort only. The cell types derived from the endoderm and ectodermof early development include epidermis, hair, nails, brain, nervoussystem, inner lining of the digestive tract, various glands, and others.Essentially, long-term cultures of normal differentiated glandular andepithelial cells, particularly those from humans, are still notavailable.

In the instance of the mammalian pancreas, until the present invention,no scientist has had the opportunity of studying, and no physician hashad the prospect of using for treatment, a cell culture of pancreaticendocrine cells that exhibited sustained cell division and the glandularproperties typical of the pancreas.

Similar to neurons, the endocrine cells of the mammalian pancreas havebeen considered to be post-mitotic, i.e., terminal, essentiallynon-dividing cells. Recent work has shown that the cells of themammalian pancreas (including those of humans) are capable of survivalin culture, but are not capable of sustained cell division. Hence, aprimary culture of the tissue cells can succeed, but due to a lack ofsufficient cell divisions of the cultured cells, passaging of theprimary culture to form serial cultures has not been possible. Althoughoccasional cells in a metaphase stage, uptake of tritiated thymidine,and other evidence of cell division have been seen in these cultures(Clark et al., Endocrinology, 126:1895 (1990); Breljie et al.,Endocrinology, 128:45 (1991)), the overall rate of cell division hasbeen considered to be below the replacement rate (that is, more, or asmany, cells die as are produced). Therefore, pancreatic endocrine cellcultures prior to the present invention were not expanded.

The inability to study pancreatic endocrine cells in culture has impededthe ability of medical science to progress in the area of pancreaticdisorders. Such disorders include diabetes mellitus, a disease thatimpairs or destroys the ability of the beta cells of the islets ofLangerhans (structures within the pancreas) to produce sufficientquantities of the hormone insulin, a hormone that serves to preventaccumulation of sugar in the bloodstream. Type I diabetes mellitus(insulin dependent, or juvenile-onset diabetes) typically requires fullhormone replacement therapy. In a second (and more common) form of thedisease, type II diabetes (sometimes referred to as late onset, orsenile diabetes), treatment often does not require insulin injectionsbecause a patient suffering with Type II diabetes may be able to controlhis/her blood sugar levels by carefully controlling food intake.However, as many as 30% of these patients also have reduced beta cellfunction and therefore are candidates for hormone replacement therapy aswell. Diabetes is not confined to humans, but has been noted in othermammals as well, such as dogs and horses.

The etiology of the diabetic disease condition is not fully understood.However, it has been noted that autoimmunity antibodies (antibodies that"mistakenly" attack bodily structures) and/or certain T lymphocytes mayhave an involvement long before clinical symptoms of diabetes emerge.Evidence in this direction relies, in part, on successful treatment ofrecently diagnosed diabetic patients with cyclosporin, animmunosuppressive drug. Such treatment has been shown to prevent orcause remission of insulin-dependent diabetes mellitus in mice (Mori etal., Diabetologia 29:244-247 (1986)), rats (Jaworski et al., DiabetesRes. 3:1-6 (1986)), and humans (Feutren et al., Lancet, 11:119-123(1986)). A clinical test to detect the presence of these humoral andcellular immunoreactions would allow the screening of individuals in apre-diabetic state, which individuals could then be prophylacticallytreated with immunosuppressive drugs.

Current treatment of individuals with clinical manifestation of diabetesattempts to emulate the role of the pancreatic beta cells in anon-diabetic individual. Individuals with normal beta cell function havetight regulation of the amount of insulin secreted into theirbloodstream. This regulation is due to a feed-back mechanism thatresides in the beta cells that ordinarily prevents surges of blood sugaroutside of the normal limits. Unless blood sugar is controlled properly,dangerous, even fatal, levels can result. Hence, treatment of a diabeticindividual involves the use of injected bovine, porcine, or cloned humaninsulin on a daily basis.

Injected insulin and diet regulation permit survival and in many cases agood quality of life for years after onset of the disease. However,there is often a gradual decline in the health of diabetics that hasbeen attributed to damage to the vascular system due to the inevitablesurges (both high and low) in the concentration of glucose in the bloodof diabetic patients. In short, diabetics treated with injected insulincannot adjust their intake of carbohydrates and injection of insulinwith sufficient precision of quantity and timing to prevent temporarysurges of glucose outside of normal limits. These surges are believed toresult in various vascular disorders that impair normal sight, kidney,and even ambulatory functions.

Both of these disease states, i.e., type I and type II diabetes,involving millions of people in the United States alone, preferablyshould be treated in a more regulated fashion. Successful transplants ofwhole isolated islets, for example, have been made in animals and inhumans. However, long term resolution of diabetic symptoms has not yetbeen achieved by this method because of a lack of persistent functioningof the grafted islets in situ. See Robertson, New England J. Med.,327:1861-1863 (1992).

For the grafts accomplished thus far in humans, one or two donatedpancreases per patient treated was required. Unfortunately only some6000 donated human pancreases become available in the United States in ayear, and many of these are needed for whole pancreas organ transplants(used when the pancreas has been removed, usually during cancersurgery). Therefore, of the millions of diabetic individuals who couldbenefit from such grafts, only a relative handful of them may be treatedgiven the current state of technology. If the supply of islet cells(including but not necessarily limited to beta cells) could be augmentedby culturing the donated islets in cell culture, expanded populationswould provide sufficient material to allow a new treatment forinsulin-dependent diabetes.

In a similar fashion, the follicle cells of the human thyroid gland arehighly specialized to respond to ambient levels of thyroid stimulatinghormone, TSH, and to synthesize thyroglobulin, a very large complexprotein that requires iodination for its activity. In response to TSHlevels, thyroglobulin is secreted as tetra-iodo and tri-iodo thyronine(T₃), which are known collectively as the thyroid hormone, thyroxine.The thyroid cells of rats have been successfully cultured in media thatallows the specialized functioning as well as the hormone dependence ofthese cells to be retained (Ambesi et al., Proc. Natl. Acad. Sci. USA,77:3455-3459 (1980)); however, analogous cell cultures of human thyroidcells have not been successfully maintained. These rat cell cultures,called FRTL and FRTL-5, and their clonal variants have become the basisfor clinical tests that seek to identify thyroid stimulating substancesin the serum of patients with suspected thyroid disease. The FRTL/FRTL5cell cultures originated from normal adult rat thyroid glands. Thesecell strains respond to thyrotropin (TSH) by releasing thyroglobulin(Tg), producing cyclic AMP (cAMP), trapping iodide, and growing. TheTSH-dependent growth in FRTL and FRTL5 cells suggested a key role of thehormone as a mitogenic factor for thyroid cells; however, not allreports have confirmed this observation (see Westermark et al., Proc.Natl. Acad. Sci. USA, 76:2022-2026 (1979); Valente et al.,Endocrinology, 112:71-79 (1983)). As to the role of cAMP, as a secondmessenger, it appears that components besides the modulation of cAMPproduction may be involved in TSH stimulatory effects (see, for example,Lombardi et al., Endocrinology, 123:1544-1552 (1988)). Whereas ingenetically engineered FRTL5 cells a pseudo-physiological rise ofintracellular cAMP level is enough to stimulate cells proliferation (Henet al., Proc. Natl. Acad. Sci. USA, 86:4785-4788 (1989)), normal thyroidcells cultured from other sources may not display the same behavior.

Other second messengers, besides cAMP, have been hypothesized to have arole in the regulation and action of thyroid cells; however, no clearempirical data support any such hypotheses (see, for example, Raspe etal., Mol. Cell. Endocrin., 81:175-183 (1991)). An important role mayalso be played by autocrine (Takahoshi et al., Endocrinology,126:7-36-7-45 (1990)) or indirect paracrine influences (Goodman andRene, Endocrinology, 121:2131-2140 (1987)). Little can be reciteddefinitively because the above-cited studies dealt with thyroid cellsfrom different animal species or from human pathological samples so thatdiscrepancies may be due to differences between species, to the variouspathological conditions, or to adaptation of the cells to the variousculture conditions used. The few studies on reportedly normal,non-transformed donor tissues have been to primary cultures, with verylittle evidence of in vitro cell proliferation (see, for example, Raspeet al., supra).

Thyroid pathologies, such as goiter, Grave's disease, Hashimoto'sdisease, adenomas, and carcinomas, involve impairment of thyroidfunction and, typically, excision of the thyroid itself. While theetiology of thyroid pathologies are not well understood, treatmentpost-excision focuses on a hormone-replacement-based therapy. If normalthyroid cells could be produced in culture in sufficient quantities,such expanded populations would provide sufficient material to allow apost-excision new treatment for these thyroid diseases.

When the thyroid gland is damaged or removed, often the parathyroidglands are also damaged or removed. While the function of the thyroidgland is rather successfully replaced by taking thyroid hormone bymouth, the parathyroid function is not easily replaced. The principalhormone product of the parathyroid gland is a protein hormone calledparathormone that is not effective if taken by mouth. Parathormoneinteracts with vitamin D and regulates mineral metabolism, particularlycalcium.

A similar situation exists with respect to the parotid glands. Theseglands are located in the angle of the jaw and are responsible forproducing much of the saliva that lubricates the oral cavity. Inparticular, three major salivary proteins are secreted by the parotidgland; namely, lumicarmine, amylase, and gustin. The absence of theparotid secretions can result in xerostomia, or dry mouth, a common,clinically disturbing but not life-threatening disorder. Xerostomiaaffects all patients following X-irradiation of the oral cavity fortreatment of oral cancers and many patients with Sjogren's syndrome.This disorder exacerbates symptoms of stomatitis, gingivitis,periodontitis, taste loss and tooth loss. Treatment of this symptom hasbeen largely unsuccessful, consisting mainly of supplying oralmoisturizers. If normal parotid cells could be produced in culture insufficient quantities, such expanded populations would providesufficient material to allow a new treatment for the xerostomicdisorder.

Other cell types have been similarly refractory in being culturedlong-term by conventional methods, particularly those of ectodermal orendodermal embryonic derivation. Among these other cell types are cellsof the olfactory neuroblasts, prostate gland, lachrymal gland,cartilage, inner ear, liver, parathyroid gland, oral mucosa, sweatglands, hair follicles, adrenal cortex, urethra, bladder, many humantumors, and others. Additionally, primary human tumor cells have notbeen susceptible to propagation in culture, including those tumor cellsof the thyroid, lung, cervix, epithelium (carcinoma), and pituitary andthyroid adenoma.

Some cell types, such as amniocytes and venous and arterial endothelium,have been cultured in vitro; however, the growth rates or the faithfulretention of differentiated functions have not proven particularlyefficacious. Growth rates of amniocytes in conventional media are suchthat the time required to grow the cells for purposes of diagnosis ofsome genetic disorders can result in providing information at a timepoint in the development of a fetus, for example, when the informationcan be acted upon only with the most dire of impact on the patient, or,perhaps, cannot be acted upon at all. Such growth rates have an economicimpact, of course, with respect to the culturing of any of theaforementioned cells. To the extent the cultured cells themselves areproducts for surgical procedures, for example, skin cells applicable toburn victims, or for production of pharmaceuticals, the existence oftechniques to cause cell culturing rates to increase results in a moreplentiful and less costly supply of those cells.

The present invention attempts to meet many of these culturing needs. Inparticular, the present invention provides a novel culturing method andmedium which are capable of producing an expanded culture of a widevariety of cells which have previously not been so cultured. Such cellsinclude pancreatic islet cells, thyroid cells, parathyroid cells,parotid cells, tumor cells, and the other cell types discussed above.The present invention further seeks to provide certain aggregates ofcells, such as pancreatic, thyroid, parathyroid and parotid cells, thathave tissue-like qualities (referred to herein as "pseudotissues"), aswell as the use of such pseudotissues for the treatment of variousdisorders, e.g., blood sugar concentration disorders, thyroiddeficiencies, parathormone deficiencies and/or mineral dyscrasia, andxerostomia in mammals. The present invention also seeks to providetechniques for the use of the cultured cells for cytotoxicity assays ofexogenous materials and to assess disease states of patients.

These and other features and advantages of the invention will be morereadily apparent upon reading the following description of preferredexemplified embodiments of the invention and upon reference to theaccompanying drawings, all of which are given by way of illustrationonly, and are not limitative of the present invention.

SUMMARY OF THE INVENTION

The present invention provides a method for producing an expandednon-transformed cell culture of a cell-type selected from the groupconsisting of glandular, neuroblast, liver, adrenal cortex, oral mucosa,cartilage, inner ear, urethra, and bladder cells, comprising the stepsof: (a) preparing the cells by mincing a tissue that comprises thecells, thereby obtaining a substructure of the tissue or free cells; (b)concentrating the substructures or cells; (c) resuspending theconcentrated substructures or cells in a culture medium capable ofsupporting sustained cell division; (d) incubating the culture; and (e)passaging the culture periodically. The culture medium preferablycomprises a basal medium and an extract of hypothalamus, pituitarygland, or placenta. The present invention further provides a method ofpreparing clonal strains, which method comprises the steps of: (a)preparing a cell culture as described above; (b) growing the cultureinto a confluent layer of cells; (c) dissociating the cells; (d)inoculating the cells into another culture vessel that contains aconditioned medium for a first plating; (e) harvesting individualcolonies of cells; (f) inoculating the colonies into another culturevessel for a second plating; and (g) passaging the resultant cellsperiodically.

The present inventive method is suitable for use with a variety ofcells, including pancreatic, thyroid, parathyroid, and parotid cells, aswell as many other types of cells.

The present invention also provides a culture medium which is useddesirably with the present inventive method. The culture mediumcomprises a basal medium and an extract of tissue or components thereofsuch that the combination does not preclude sustained cell division bycultured cells that are derived from exocrine or endocrine glands. Thebasal medium is preferably Coon's Modified F12 Medium, while the tissueis preferably selected from the group consisting of hypothalamus,pituitary gland, and placenta.

The present invention additional provides expanded cell cultures ofpancreatic endocrine cells, thyroid cells, and parotid cells and methodsof using such cell cultures in diagnostic assays and in therapeutictreatments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that shows the accumulation of insulin and C-peptidein the medium of HPSL-6 cultured cells without glucose challenge.

FIG. 2 is a graph that shows the results of glucose stimulation of theHPSL-8 strain of pancreatic cells, in terms of cell growth, productionof insulin, and C-peptide.

FIGS. 3A-3D are graphs that show the effects of modifications of Coon's4506.07 medium on hormone secretion by HPSL-8 cells following glucosechallenge.

FIGS. 4A-4D are graphs that show the effects of modifications of Coon's4506.07 medium without added insulin on the rate of hormone secretion byHPSL-8 cells following glucose challenge.

FIGS. 5A-5D are graphs that show the effects of modifications of Coon's4506.07 medium with added insulin on hormone secretion rate by HPSL-8.

FIGS. 6A-6B are graphs that show the regulation of blood sugar levels indiabetic mice that have received grafts of pseudotissues comprising ofpseudoislets or suspended cells.

FIG. 7 is a graph that shows the effect of TSH with and without insulinon the growth of thyroid culture cells.

FIG. 8 is a graph that shows the TSH-stimulated dose-dependent increaseof cAMP accumulation in FRTL or NHTB-2K cells.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention is provided to aidthose skilled in the art in practicing the present invention. Thisdetailed description should not be construed to limit the presentinvention, as modifications and variations in the embodiments hereindiscussed may be made by those of ordinary skill in the art withoutdeparting from the spirit or scope of the present inventive discovery.

The present invention provides for a method for producing an expandednon-transformed cell culture of a cell-type selected from the groupconsisting of glandular, neuroblast, liver, adrenal cortex, oral mucosa,cartilage, inner ear, urethra, and bladder cells, comprising the stepsof: (1) preparing said cells by mincing a tissue that comprises thecells, thereby obtaining cells or substructures of the tissue; (2)concentrating the cells or substructures; (3) resuspending theconcentrated cells or substructures in a culture medium capable ofsupporting sustained cell division; (4) incubating the culture; and (5)passaging the culture.

The cell-types subjected to this procedure are derived from varioustissues, can be of human origin or that of any other mammal, and may beof any suitable source, such as a whole pancreas, parotid gland, thyroidgland, parathyroid gland, prostate gland, lachrymal gland, cartilage,kidney, inner ear, liver, parathyroid gland, oral mucosa, sweat gland,hair follicle, adrenal cortex, urethra, and bladder, or portions ormultiples thereof. The tissue is prepared using any suitable method,such as by gently teasing apart the excised tissue or by digestion ofexcised tissue with collagenase via, for example, perfusion through aduct or simple incubation of, for example, teased tissue in acollagenase-containing buffer of suitable pH and tonic strength. Theprepared tissue then is concentrated using suitable methods andmaterials, such as centrifugation through ficol gradients forconcentration (and partial purification). The concentrated tissue thenis resuspended into any suitable vessel, such as tissue cultureglassware or plasticware. The resuspended material may include wholesubstructures of the tissue, cells and clusters of cells. For example,such substructures may include islets and ducts in the case ofpancreatic tissue and follicles in the case of thyroid tissue.

The initial culture of resuspended tissue cells is a primary culture. Inthe initial culturing of the primary culture, the cells attach andspread on the surface of a suitable culture vessel with concomitant celldivision. Subsequent to the initial culture, and usually after therealization of a monolayer of cells in the culture vessel, seriallypropagated secondary and subsequent cultures are prepared bydissociating the cells of the primary culture and diluting the initialculture or its succeeding cultures into fresh culture vessels, aprocedure known in the art as passaging. Such passaging results in anexpanded culture of cells of the originating tissue. The cell culture ispassaged at suitable intervals, such as about once a week or after abouttwo to about three cell divisions of the cultured cells. Longerintervals of two to three weeks or shorter intervals of two to threedays would suffice also. For passaging the cell cultures, a dilution ofthe cultured cells at a ratio of from about 1:2 to about 1:100 is used.Preferably, a ratio of from about 1:4 to about 1:50 is used. Morepreferably, a ratio of from about 1:4 to about 1:6 is used.

The concentrated prepared tissue, which may be in the form of free cellsand/or clumps (where the clumps may constitute ordered substructures ofthe tissue) is resuspended at any suitable initial cell or presumptivecell density. Suitable cell densities range from about 100 cells toabout 1000 cells per square centimeter of surface area of the culturevessel. Such cell densities for initial plating are best illustrated byidentifying such parameters for specific cell systems, as follows.

For culturing pancreatic tissue cells, the concentrated islets areresuspended at any suitable initial islet density, such as at an initialislet density of from about 1 to about 700 islets per square centimeterof surface area of the culture vessel, which is equivalent to an initialislet density of from about 100 to about 50,000 islets per standard 100mm diameter petri dish. In a preferred embodiment, the concentratedislets are resuspended at a density of from about 1 to about 70 isletsper square centimeter of surface area of the culture vessel, which isequivalent to an initial islet density of from about 100 to about 5000islets per standard 100 mm diameter petri dish. In a more preferredembodiment, the concentrated islets are resuspended at a density of fromabout 1 to about 7 islets per square centimeter of surface area of theculture vessel, which is equivalent to an initial islet density of fromabout 100 to about 500 islets per standard 100 mm diameter petri dish.In another more preferred embodiment of this method, the concentratedislets are resuspended at a density of from about 3 to about 7 isletsper square centimeter of surface area of the culture vessel, which isequivalent to an initial islet density of from about 250 to about 500islets per standard 100 mm diameter petri dish.

For culturing thyroid tissue cells, the concentrated cells and fragmentsof follicles are resuspended at any suitable initial density, such as atan initial cell density of from about 10⁴ cells to about 10⁶ cells per100 mm diameter petri dish (Falcon, Becton Dickinson, Lincoln Park,N.J.). In a preferred embodiment, the concentrated thyroid cells areresuspended at a density of from about 6×10⁴ cells to about 5×10⁵ cellsper 100 mm diameter petri dish. In a more preferred embodiment, theconcentrated thyroid cells are resuspended at a density of about 8×10⁴cells to about 3×10⁵ cells per 100 mm diameter petri dish.

The method for producing an expanded cell culture depends on the use ofa culture medium that comprises a suitable basal medium and a suitableextract of a suitable tissue, the combination of which is designed notto preclude sustained cell division by the cultured cells derived fromthe aforementioned tissues, including exocrine and endocrine glands.Serum or components derived therefrom typically are also included in themixture. Components of the tissue extract may be used in place of thecrude or partially purified tissue extract.

Basal media that may be used include those commercially available fromSigma Chemical Co., Life Technologies, Inc., or BioWhittaker Co. Anybasal medium may be used provided that at least magnesium ion, calciumion, zinc ion, bicarbonate ion, potassium ion, and sugar levels can bemanipulated to a lower or higher concentration in the resultant medium;in particular, the magnesium ion, calcium ion, bicarbonate ion, andD-glucose levels are required at a lower concentration, zinc ion isrequired at the same or higher concentration, and potassium ion isrequired at the same or lower concentration than is usual in standardbasal media.

Preferred levels of magnesium ion, as contributed by suitable magnesiumsalts, such as MgSO₄ •7H₂ O and MgCl₂ •6H₂ O, are between 60 and 240mg/L; more preferred levels of magnesium salts are between 100 and 150mg/L. Preferred levels of calcium ion, as contributed by suitablecalcium salts, such as CaCl₂ •2H₂ O, are between 25 and 200 mg/L; morepreferred levels of calcium ion are between 40 and 125 mg/L. Preferredlevels of zinc ion, as contributed by suitable zinc salts, such as ZnSO₄•7H₂ O, are between 0.1 and 0.5 mg/L; more preferred levels of zinc ionare between 0.12 and 0.40 mg/L; yet more preferred levels of zinc ionare between 0.15 and 0.20 mg/L. Preferred levels of ascorbic acid arebetween 30 and 125 mg/L; more preferred levels of ascorbic acid arebetween 40 and 100 mg/L. Preferred levels of bicarbonate ion, ascontributed by suitable bicarbonate salts, such as sodium bicarbonate,are between 175 and 700 mg/L; more preferred levels of bicarbonate ionare between 300 and 400 mg/L. Preferred levels of potassium ion, ascontributed by suitable potassium salts, such as potassium chloride, arebetween 100 and 400 mg/L; preferred levels of potassium ion are between200 and 325 mg/L; most preferred levels of potassium ion are between 210and 250 mg/L. Preferred levels of sugar, as contributed by a suitablesugar, such as D-glucose, are between 400 and 1800 mg/L; more preferredlevels of sugar are between 600 and 1200 mg/L; most preferred levels ofsugar are between 800 and 1000 mg/L. Preferred levels of human placentallactogen are between 3 and 15 μg/ml; more preferred levels of humanplacental lactogen are between 4 and 13 μg/ml; most preferred levels ofhuman placental lactogen are between 8 and 12 μg/ml. Preferred levels ofinsulin, as contributed by a suitable naturally-isolated,clonally-derived, or synthesized insulin, such as isolated bovinesodium-insulin, are between 50 and 20,000 ng/ml; more preferred levelsof insulin are between 100 and 10,000 ng/ml; most preferred levels ofinsulin are between 500 and 5,000 ng/ml.

One basal medium that can be used preferably is Coon's Modified F12Medium (Coon et al., Proc. Natl. Acad. Sci. U.S.A., 86, 1703 (1989)),which is available from BioWhittaker Co., Walkerville, Md., or preparedaccording to the formula provided in Example 1.

The tissue extracts that may be used to prepare the present inventiveculture medium include any suitable tissue that contains growth factors.Such tissues preferably include at least one of hypothalamus, pituitarygland, and placenta. As noted above, suitable components of a tissueextract, such as a partially or wholly purified solution containingsuitable growth factors, or synthetic varieties thereof, may be used inplace of the whole tissue extract, such as human placental lactogen inplace of human placental extract. Such suitable components of a tissueextract may also be used in addition to the whole tissue extract, suchas human placental lactogen plus human placental extract.

Serum is used at levels lower than those typically used by practitionersof the relevant art. For example, typical cell culture media use 10% to20% fetal bovine serum, whereas the medium of the present invention usesless than 10% serum and generally from about 2% to about 6% serum. Thepreferred concentration of serum in the medium of the present inventionis from about 3% to about 5%. The more preferred concentration of serumin the medium of the present invention is about 4%. Sources of serainclude bovine fetuses and new born calves, and equine, porcine, ovine,and human fetuses and adults. Preferably, bovine fetal serum is used.Also, suitable components of sera, such as a partially or whollypurified solution derived from sera containing suitable growth factors,may be used in place of the whole serum. Suitable growth factorsprovided in serum may also be produced synthetically and may therebyreplace the need for serum.

The preferred culture media are Coon's 4506.035 and Coon's 4506.07media, as defined in Example 1. Coon's 4506.035 and Coon's 4506.07 mediacontain lower calcium ion (Ca⁺⁺) concentration, lower added serumconcentration (4% versus 10-20% fetal bovine serum), and a relativelyhigh concentration of growth factors as provided by the tissue extractcomponents and human placental lactogen, as compared to Coon's modifiedF12 Medium from which these media can be prepared. Although fibroblaststypically overgrow cultures of glandular cells, such fibroblast cells,which are commonly co-purified with islet cells, for example, do notovergrow cultures in Coon's 4506.035 or 4506.07 medium when they aremaintained in continuous, serial passage. Using Coon's 4506.07 medium,for example, fibroblasts grow 25-50% slower than in a conventionalmedium, such as 10% Fetal Calf Serum in Dulbeco's Modified Eagle'sMedium.

The mass cultures of islets grown in Coon's 4506.035 or 4506.07 medium,in effect, become enrichment cultures for the endocrine cells of theislets. This failure of endogenous fibroblasts to overgrow and crowd outthe functional endocrine cells is important in the success of culturesof pancreatic endocrine cells of the present invention as compared toearlier attempts to grow these cells. Similarly, this feature isimportant for growth of the other mentioned cell types.

The present invention also provides for a method for preparing clonalstrains from each of the cell cultures discussed herein, including, forexample, the pancreatic endocrine cell culture discussed herein,comprising the steps of: (1) preparing a cell culture according to theprocedure outlined above; (2) growing the culture; (3) dissociating thecells; (4) inoculating the cells into another culture vessel for a firstplating; (5) harvesting individual colonies of cells; (6) inoculatingthe colonies into another culture vessel for a second plating; and (7)passaging resultant clonal cell strains periodically.

A culture of cells may be used for preparing clonal strains upon havinggrown into a confluent layer of cells, or the culture may be used priorto having reached confluence. Dissociation may be effected using anysuitable means, such as by trypsin or some other proteolytic treatment.Any suitable density of cells per square centimeter of surface area of aculture vessel can be used for the first plating, such that the growthof individually isolable colonies is promoted. Preferably, between about3 and about 150 cells per square centimeter of surface area of a culturevessel is used; more preferably, between about 7 and about 70 cells persquare centimeter is used.

For the preparation of clonal strains, a conditioned medium is requiredfor the first plating described immediately above, wherein the mediummay be conditioned homologously (i.e., by the same type of cells thatare to be cloned) or heterologously (i.e., by cells other than the typethat is to be cloned). The conditioned medium can be prepared by thesteps of: (1) incubating cultured cells, as prepared according to theprocedure described herein; (2) harvesting the medium; and (3) sterilefiltering the resultant conditioned medium.

The cell density used for preparation of the conditioned medium canrange from very few cells per square centimeter of surface area of aculture vessel to near confluence. The length of time of incubationrequired is inversely dependent on the cell density. In essence, asuitable concentration of excreted cell products from the cells formsnecessary ingredients of conditioned medium, which concentration isreached more quickly with the greater number of cells incubated per unitvolume of culture medium. It is necessary that the cells grow; thus anydensity that is less than confluence will suffice to prepare theconditioned medium. Preferably, the cell density ranges from about 5×10³to about 5×10⁴ cells per square centimeter of surface area of a culturevessel, where the period of incubation ranges from about 18 hours toabout 24 hours. In accordance with the aforementioned inverserelationship, if fewer cells are incubated, then a longer period ofincubation is required; if more cells are plated, then a shorter periodof incubation is required.

As regards the amount of culture medium in which the cells areincubated, the culture vessel may contain any suitable amount of culturemedium and preferably should contain from about two to about fourmilliliters per 10⁶ cells. Preferred culture media include Coon's4506.035 and Coon's 4506.07.

Harvesting of the conditioned medium is undertaken using any suitablemeans, such as pouring off or aspirating the medium into a suitablecontainer, such as a flask. Sterile filtering of the harvested medium isundertaken using any suitable means, such as passing the medium througha suitable ultrafiltration membrane while under pressure. Alternatively,the medium may be filtered in a diafiltration process known to the art,also using membrane filters.

For inoculation of the first plating in the procedure for preparation ofclonal strains, the sterile filtered conditioned medium is diluted tomake it suitable for promoting growth of the inoculum. Preferably, threeto five parts of the conditioned medium are diluted with one to threeparts of a suitable culture medium. More preferably, about two parts ofthe conditioned medium are diluted with about one part of a suitableculture medium.

Individual colonies that form in the first plating are harvested after asuitable number of population doublings, which colonies thereforecomprise a suitable number of cells. Preferably, the colonies areharvested after from about seven to about fifteen population doublings,at which point the colonies comprise from about 128 cells to about32,000 cells. More preferably, the colonies are harvested after fromabout nine to about twelve population doublings, at which point thecolonies comprise from about 500 to about 4,000 cells.

Inoculation for the second plating may be accomplished using anysuitable starting cell density. The number of cells to be used islimited by the amount included in a selected colony; thus the platingdensity here is altered by changing the size of culture vessel and theamount of culture medium. The densities are similar to those preferablyused for production of conditioned medium. Standard culture vessels of30 mm diameter petri plates or microtiter plates (which have 5 mmdiameter wells), for example, may be used to provide the appropriateratio of surface area to number of cells in an inoculum.

Preferably from about one to about three parts of the conditioned mediumis diluted with from about one to about three parts of the culturemedium for feeding the cells of the first plating and the cells of thesecond plating. More preferably, about one part of the conditionedmedium is diluted with about one part of the culture medium for feedingthe cells of the first plating and the cells of the second plating.

Passaging of the cloned culture is accomplished with the same method asused to passage the primary and serially propagated cultures describedabove. The medium used when passaging the cloned strain of cells may beany suitable one as described above for the initial plating of isletcell preparations for primary cultures. Although conditioned medium maybe used for passaging the cloned culture, a fresh medium is preferred.

An objective of the procedures described hereinabove is to isolatediploid, non-transformed cell cultures of various cell types that arecapable of sustained cell division, wherein each culture contains asingle cell type or related cell types. This objective has beenaccomplished using pancreatic tissue, thyroid tissue, parathyroidtissue, and parotid tissue, all of human origin, although such tissuesderived from other mammals, such as dogs or horses, could be used aswell. Such cultures also gave rise to cultures of cells that werederived from a single progenitor cell. Hence, pancreatic endocrine andduct, thyroid, parathyroid and parotid cells are not post-mitotic, atleast when stimulated using the present inventive culture medium. Thecells in culture remained diploid and retained other characteristics(presented below in Examples 5, 12 and 13, for example) that indicatethat the pancreatic endocrine and duct, thyroid, and parotid cellcultures, for example, of the present invention were not transformed toa premalignant state. It has been also noted that cultures that werestarted with partially purified pancreatic islets, for example, composedof not only alpha, beta, delta, and duct pancreatic cells but alsofibroblasts, macrophages, etc., were populated preponderantly bypancreatic endocrine cells using the medium of the present invention.Apparently, the medium of the present invention selects in favor of thepancreatic endocrine cells and against the other cells that areapparently co-purified with pancreatic islets.

As described in detail below, the pancreatic endocrine cell cultures ofthe present invention can be used as the basis for assays whose purposeis to identify cytotoxic agents of any source that are directed at isletcells. Similarly, thyroid cells, parotid cells, and other cells may beused analogously. Cytotoxicity, in general, is measured by exposing cellcultures to dilutions of a suspected toxic agent and, at some latertime, assessing the number of killed or dead cells. With the advent offunctionally differentiated human cells, a novel and more subtle assayis possible. In addition to monitoring dead cells, one can quantitatethe ability of the suspected toxic substance(s) to interfere with normalphysiological functions, e.g., to interfere with the ability of humanbeta cells in culture to respond to changes in the ambient glucoseconcentration by secreting insulin. An assay of this kind allowsevidence of non-lethal but nevertheless toxic responses that mightinterfere with either the glucose-sensing process or theinsulin-secretion process including the pre-insulin processing stepevidenced by C-peptide release. Changes in the shape of the glucoseconcentration versus insulin secretion curves can indicate suchimpairment of normal physiological function, and measurements usingestablished analytical procedures, like RIA, can quantitate both insulinsecretion and C-peptide release into the medium.

Accordingly, the cultured cells of the present invention, which exhibitthe characteristics of normal human cells, may be used in tests designedto detect the presence of cytotoxic agents of any kind, such as are usedin the food industry, pharmaceutical industry, cosmetic industry, andother industries. In the area of medical diagnostics, such tests includeclinical assays designed to detect certain autoantibodies orT-lymphocytes in the blood or tissues of patients with diabetes orpossible diabetes. Such autoantibodies or T lymphocytes would beidentified by their ability to interact with the cultured cells or tofoster cytotoxic reactions in them or upon them.

This diagnostic assay comprises exposing a diploid cell culture ofpancreatic endocrine cells that is capable of cell division to achemical or sample of bodily fluid and assessing the effects of theexposure of the cells. The diploid cell culture is as described above,and may originate from any mammal; preferably the cell cultureoriginates from a human. By exposure, it is intended that a testedchemical is put into solution and then diluted into the culture mediumin which the test cells are incubating. Similarly, a suitable bodilyfluid, such as blood serum, spinal fluid, mucous, etc., would be testedby diluting it into the culture medium in which the test cells areincubating. Serial dilutions of the test samples and positive andnegative controls would also be included in this procedure. Assessmentof the effect of the chemical or bodily fluid diluted into the culturemedium of a test culture can be accomplished using any suitable means,such as by tracking vital signs of the culture using methods known inthe art. Such trackable vital signs include population doubling time andmetabolic rate. Preferably, cultured pancreatic beta cells challenged byinclusion of a suspected cytotoxin in the culture medium are assessedfor response to changes in the ambient glucose concentration usingmethods known in the art. The primary response for assessment isinsulin-secretion and the prior step of processing of pre-insulin andthe resultant release of C-peptides.

The donated human pancreas cells used in the diagnostic test assay forautoantibodies and cytotoxic T-cells are taken preferably fromindividuals with the HLA markers associated with high incidences ofIDDM. Among these markers are the HLA Class II antigens DR-3, DR-4,DW-3, DW-4 and B-8, B-15, which are associated with greatly increasedrisk of developing IDDM.

The donated human pancreas cells used in preparing pseudotissues forgrafting for the purpose of regulating blood sugar levels are preferablytaken from individuals with the HLA markers that are rarely if everassociated with development of IDDM. Among these are the HLA Class IIantigens DR-5, DR-2, BW-2, BW-3, BW-8 and A-11.

Similarly, another preferred aspect of the present invention relates tocultured thyroid cells challenged by inclusion of a suspected cytotoxinin the culture medium, the toxicity of which is assessed by response ofthe cultured cells to ambient TSH concentrations using methods known inthe art. The primary response for assessment can be cAMP production andiodide uptake. Analogous cytoxicity tests involving other cell typesthat are newly culturable by use of the present inventive cultures arealso aspects of the present invention.

The present invention also concerns therapeutic methods involving theuse of the present inventive cultures. For example, the presentinvention provides a method of altering blood sugar levels comprisingadministering to a mammal a cell culture of pancreatic endocrine cells.The cell culture used for altering blood sugar levels may be a primarycell culture of pancreatic endocrine cells, or a serially passagedculture thereof. Preparation of such a cell culture is as describedhereinabove. The cell culture used may also be a clonal cell culture ofpancreatic endocrine cells, preparation of which is as describedhereinabove also. The cultured pancreatic endocrine cells of the presentinvention include beta cells that secrete insulin in response to glucoseconcentration.

The method of altering blood sugar levels may be accomplished usingcultured pancreatic endocrine cells in a tissue-like form. Such culturedpancreatic endocrine cells, either as individual beta cells or incombination with other cell types, can form coherent aggregatesspontaneously or by culturing techniques known in the art. Such coherentaggregates are termed "pseudoislets"herein. Preferably, pseudoislets areembedded in a suitable biocompatible matrix, such as collagen, usingmethods known in the art. The cultured pancreatic endocrine cells alsomay be formed into coherent aggregates by co-incubation with a suitablebiocompatible material, such as collagen, whereby the cells are in theform of free suspensions prior to the co-incubation. The coherentaggregate of cells formed by either method is termed a "pseudotissue."Pseudotissues form a biologically compatible graft that can be implantedinto a mammal, and therein function to alter blood sugar levels.

Primary, secondary and subsequent, or clonal cultures of pancreaticendocrine cells, or combinations thereof prepared according to themethods described herein, and exemplified below, may be used in suchpseudotissues. The method involves grafting pancreatic endocrine cellsas a pseudotissue, for example, into a mammal where the pseudotissuebecomes vascularized and responds to the blood glucose levels in thehost mammal by secreting insulin when the blood glucose levels attain asufficiently high level. Vascularization of the pseudotissue appears tobe important in that in those experiments where the pseudotissue did notbecome vascularized, blood sugar levels were not regulated. Similarly,delayed vascularization of a pseudotissue appeared to impair the abilityof the pseudotissue to regulate blood sugar levels. A practicaldemonstration of successful pseudotissues according to the presentinvention is illustrated in Example 10 below in an experimental diabeticmouse system. However, the same approach can be used to treat aberrantblood sugar levels in other mammals as well, most particularly humans,dogs, and horses.

The present invention also concerns a method of providing thyroidhormones or parathormone comprising administering to a mammal a cellculture of thyroid or parathyroid cells, respectively. The cell cultureused for providing thyroid hormones or parathormone may be a primarycell culture of thyroid or parathryoid cells, or a serially passagedculture thereof. Preparation of such a cell culture is as describedhereinabove. The cell culture used may also be a clonal cell culture ofthyroid or parathyroid cells, preparation of which is as describedhereinabove also. The cultured thyroid or parathyroid cells of thepresent invention include thyroid follicle cells that secrete thyroidhormones in response to TSH concentration and parathyroid tissue thatsecretes parathormone.

The method of providing thyroid hormones or parathormone may beaccomplished using cultures of appropriate cells derived from therespective glands that are formed into a tissue-like form. Such culturedgland cells, either as individual follicle cells, in combination withother cell types, or dissociated gland cells, can form coherentaggregates spontaneously or by culturing techniques known in the art.Such coherent aggregates are termed "thyroid pseudotissue" or"parathyroid pseudotissue," as appropriate, herein. Preferably, suchpseudotissues are embedded in a suitable biocompatible matrix, such ascollagen, using methods known in the art. The cultured gland cells alsomay be formed into coherent aggregates by co-incubation with a suitablebiocompatible material, such as collagen, whereby the cells are in theform of free suspensions prior to the co-incubation. The coherentaggregate of cells formed by either method is termed a "thyroidpseudotissue" or a "parathyroid pseudotissue," as appropriate. Thyroidor parathyroid pseudotissues form a biologically compatible graft thatcan be implanted into a mammal, and therein function to provide thyroidhormone or parathormone, depending on the derivation of the cells thatform the pseudotissue used.

Primary, secondary and subsequent, or clonal cultures of thyroid orparathyroid cells, or combinations of primary, secondary and subsequent,or clonal cultures prepared according to the methods described herein,and exemplified below, may be used in such thyroid or parathyroidpseudotissues. The method involves grafting the appropriate cells as athyroid pseudotissue, for example, into a mammal where the thyroidpseudotissue becomes vascularized and responds to the blood TSH levelsin the host mammal by secreting thyroid hormones, producing cAMP, andintaking iodide when the blood TSH levels attain a sufficiently highlevel. Similarly, a parathyroid pseudotissue may be grafted into amammal where the pseudotissue becomes vascularized and responds to bloodcalcium levels, for example, by secreting parathormone.

Collagen is ordinarily extracted under acid conditions and, ifsubsequently neutralized, remains liquid at 4° C. At 37° C., neutralizedsolutions of collagen irreversibly form into a gel. Thus, if a cellsuspension is made in a collagen solution at 4° C. and then incubated at37° C., the cells will become embedded in a gel. At high densities (1:1collagen:cells), one variety of pseudotissue is formed that can be castand formed into shapes suitable for implantation at various sites (e.g.,as a sheet for implantation subcutaneously). Alternatively, the cellsmay first be allowed to reaggregate into clusters of from 25-250 cells(pseudoislets), and then these clusters in turn may be embedded in acollagen gel as above, thereby forming another variety of pseudotissue.In either case, the use of a collagen gel is known to promotevascularization and healing of graft cells into the tissues of the hostanimal.

The following examples are illustrative of the preparation and use ofthe products and methods of the present invention. As such, thefollowing examples further illustrate the present invention but, ofcourse, should not be construed as in any manner limiting its scope.

EXAMPLE 1

This example illustrates the preparation of media suitable for growingtissue cells in culture. In particular, Coon's 4506.07 and 4506.035media are described.

Growth media in accordance with the present invention were prepared bycombining Coon's Modified F12 Medium containing no added calcium andreduced KCl with mixtures of tissue extracts. The formula of Coon'sModified F12 Medium is recited below.

The tissue extracts were made as described in Coon et al., Proc. Natl.Acad. Sci. U.S.A., 86, 1703 (1989). Frozen tissue was homogenized in aWaring blender with a two-fold dilution (1:2 wt/vol) aqueous HEPESbuffer (200mM adjusted to pH 7.2 with NaOH). The tissue homogenate thusformed was refrigerated for 30 minutes in a refrigerator (4° C.),remixed by a short period of blending, and refrigerated for anadditional 30 minutes. After remixing as described, two refrigerated(≦6° C.) centrifugations were performed. First, a low speedcentrifugation for 1 hour at approximately 30,000×g of the homogenatewas carried out. The supernatant fluid was then decanted and immediatelyrecentrifuged for 1 hour at approximately 150,000×g in anultracentrifuge. When the supernatant fluid (the extract) was aspiratedfrom the centrifuge tubes, the most dense material at the bottom of thetube was carefully prevented from contaminating the final productbecause this material contains substances that make sterile filtrationvery difficult. The extracts were frozen quickly by submersing partlyfilled plastic tubes in liquid nitrogen. Extracts were made in this wayof bovine hypothalami and whole pituitary glands. Human placentae werealso extracted using this method; however, because the placenta is sucha tough and fibrotic tissue, it was necessary first to grind or cut upthe tissue before the homogenization step.

After the preparation of the ingredients was complete, they werecombined in a manner known in the art to provide media having thebelow-indicated final concentrations of each component. Empiricalobservation demonstrated that certain of the ingredients of the Coon's4506.035 and 4506.07 Media could be varied within certain ranges, orhave alternative concentrations, as indicated in the table below. Oneingredient, Na-Insulin, was determined empirically to be useful over arange of concentrations, as indicated in the table; the usualconcentration used for the present inventive growth medium is indicatedin a footnote below the chart. Unless otherwise noted, all values are inmg/L units.

    ______________________________________                                                    Coon's.sup.▴                                                            Coon's   Coon's                                                      mF12     4506.035 4506.07                                         ______________________________________                                        L-Arginine HCl                                                                              420        420      420                                         L-Histidine Hcl                                                                             42         42       42                                          L-Isoleucine  8          8        8                                           L-Leucine     26         26       26                                          L-Lysine Hcl  73         73       73                                          Glycine       16         16       16                                          L-Methionine  9          9        9                                           L-Phenylalanine                                                                             10         10       10                                          L-Serine      21         21       21                                          L-Threonine   24         24       24                                          L-Tryptophan  4          4        4                                           L-Tyrosine    11         11       11                                          L-Valine      23.4       23.4     23.4                                        L-Cysteine    0          0        0                                           L-Cystine.HCl.H.sub.2 O                                                                     70         70       70                                          L-Asparagine.H.sub.2 O                                                                      30         30       30                                          L-Proline     70         70       70                                          L-Alanine     18         18       18                                          L-Aspartic acid                                                                             26         26       26                                          L-Glutamic acid                                                                             30         30       30                                          Sodium pyruvate                                                                             220        220      220                                         Putrescine.2HCl                                                                             0.30       0.3      0.3                                         Biotin        0.07       0.07     0.07                                        D-Ca-Pantothenate                                                                           0.50       0.5      0.5                                         Niacinamide   0.04       0.04     0.04                                        Linoleic acid 0.09       0.09     0.09                                        Pyridoxine.HCl                                                                              0.06       0.06     0.06                                        Thiamine.HCl  0.285      0.285    0.285                                       Riboflavin    0.04       0.04     0.04                                        Folic acid    1          1        1                                           Vitamin B-12  1          1        1                                           Thioctic acid 0.2        0.2      0.2                                         myo-Inositol  36         36       36                                          Ascorbic acid 45         45-100   45-100                                      Choline.HCl   13.8       13.8     13.8                                        Thymidine     0.7        0.7      0.7                                         Hypoxanthine  4          4        4                                           NaCl          7530       7530     7530                                        KCl           305        230      230                                         Na.sub.2 HPO.sub.4.7H.sub.2 O                                                               250        250      250                                         KH.sub.2 PO.sub.4                                                                           68         68       68                                          MgSO.sub.4.7H.sub.2 O                                                                       104        60       60                                          MgCl.sub.2.6H.sub.2 O                                                                       106        60       60                                          CaCl.sub.2.2H.sub.2 O                                                                       165        53       105                                         CuSO.sub.4.5H.sub.2 O                                                                       0.002      0.002    0.002                                       ZnSO.sub.4.7H.sub.2 O                                                                       0.15       0.15     0.15                                        FeSO.sub.4.7H.sub.2 O                                                                       0.80       0.80     0.80                                        D-Glucose     2000       1000     1000                                        NaHCO.sub.3   2500       350      350                                         L-Glutamine   292        292      292                                         Phenol red    1.25       1.25     1.25                                        Na-Insulin (bovine)                                                                         0          100 to   100 to                                                               10,000   10,000                                                               ng/ml*   ng/ml*                                      Transferrin (bovine)                                                                        0          5 μg/ml                                                                             5 μg/ml                                  Tri-iodothyronine (T.sub.3)                                                                 0          40 pg/ml 40 pg/ml                                    Selenous acid 0          2.5 ng/ml                                                                              2.5 ng/ml                                   Hydrocortisone                                                                              0          3.5 ng/ml                                                                              3.5 ng/ml                                   Gentamycin SO.sub.4                                                                         0          50 μg/ml.sup.                                                         50 μg/ml.sup.              Fetal CaIf Serum                                                                            0          40 ml/L  40 ml/L                                     Pituitary Extract                                                                           0          50 μg/ml.sup.♦                                                        50 μg/ml.sup.♦             Hypothalamus  0          115 μg/ml.sup.♦                                                       115 μg/ml.sup.♦            Extract                                                                       Human Placenta                                                                              0          50 μg/ml.sup.♦                                                        50 μg/ml.sup.♦             Extract                                                                       Human Placenta                                                                              0          10 μg/ml                                                                            10 μg/ml                                 Lactogen                                                                      ______________________________________                                         *Usual concentration is 300 ng/ml.                                            ▴Coon's modified F12 medium.                                   Optional component (antibiotic).                                Either Human Placental Extract or Human Placental Lactogen were used; not     both.                                                                         ♦Based on protein content of extract.                           Usual concentration is 45 mg/L.                                          

EXAMPLE 2

This example illustrates the preparation of partially purified islets ofLangerhans from explanted pancreatic tissue, and the primary culturingof partially purified islets to provide mass cultures of pancreaticendocrine cells.

Pancreases or portions thereof were obtained from adult human donorsbelieved to have had normal pancreatic function. The pancreatic tissueused herein was received from a total of 11 adult human patients, bothmales and females, and collected by two medical transplant groups basedin Milan, Italy and St. Louis, Mo. No differences were noted in theculturing or glandular characteristics of cultured cells derived fromthese sources.

Partially purified pancreatic islets of Langerhans of the Milan and St.Louis pancreatic tissues were provided by Drs. Valerio Di Carlo, GuidoPozza, and Carlo Socci, San Raffaele Hospital, Milan, Italy and by Drs.Scharp and Lacy of Washington University Medical School, BarnesHospital, St. Louis, Mo. Established methods were used to prepare theislets, including mincing the pancreatic tissue or perfusing the wholepancreas via the common duct with a solution of collagenase, and finalseparation on ficol step gradients to produce concentrated populationsof islets largely purified from other pancreatic tissue components. Inthis manner approximately 300,000 islets were prepared of which5,000-10,000 were used.

The isolated islets were plated one day after preparation directly intoa culturing vessel, which was tissue culture grade glassware orplasticware (Falcon and Corning brands were used with equal success),using Coon's 4506.035 or Coon's 4506.07 Medium (described in Example 1).Between 300 and 500 islets were placed into 35 each standard 100 mmdiameter petri plate, where they attached to the surface. The cells ofthe islets grew and spread out on the culture vessel surface, and aftera period of time (usually two to three weeks), they were trypsinized instandard fashion (with or without EDTA or EGTA chelation) to dissociatethem from the vessel surface and from each other.

EXAMPLE 3

This example illustrates maintenance of the mass pancreatic endocrinecell cultures by passaging.

The mass primary cultures produced by the many islets (100 to 500 isletsper standard 100 mm diameter petri plate) growing and spreading in thesame culture vessel were maintained in log phase growth by trypsinizingthem and diluting them 4 to 6 fold into new vessels weekly. Long termserial passages grew at a rate of about 2.5 population doublings perweek. The cells so cultured were observed to become enriched forendocrine cells of the islets over other cells that were co-purifiedwith the islets, such as fibroblasts and capillary endothelial cells.After 10 passages, for example, HPSL-8 cultures included very few or nofibroblasts (judging by cell morphology). Moreover, no evidence ofcapillary endothelial cells was found using an indirectimmunofluorescence assay for factor-VIII-related-antigen.

EXAMPLE 4

This example illustrates preparation of conditioned medium.

Both primary cultures and the mass cultures made by serial passage ofthe primary cultures described in Example 2 above were used separatelyto produce a derivative medium, called conditioned medium (CM). CM wasused for cloning the cells from the primary culture plates, as well asfrom the serially propagated passage plates. CM was made by adding toculture vessels containing about 5×10⁴ cells per square centimeter (cm²)of vessel surface area and sufficient Coon's 4506.07 medium to result inabout 2.75 ml medium per 10⁶ cells, then incubating them for 20-25hours, harvesting the medium and sterile filtering (using a Millipore0.22 μm membrane, or equivalent) immediately before use.

EXAMPLE 5

This example illustrates a method for preparation of a clonal strain ofcultured pancreatic endocrine cells and sets forth the results of ananalysis of the cloned strains.

Mass cultures of pancreatic endocrine cells prepared according toExample 2 were used as a source of pancreatic endocrine cells, whichwere plated in a sequence of two platings. Conditioned media, asprepared according to Example 4, were diluted two parts CM with 1 partfresh Coon's 4506.07 Medium for the first plating. Suspensions offreshly trypsinized cells were plated at densities of 500, 1000, 2000,and 5000 cells per standard 100 mm diameter plastic petri plate.Thereafter, the clone cultures were fed twice weekly with freshlyprepared CM diluted 1:1 with fresh Coon's 4506.07 Medium.

Well isolated, circular, homogeneous colonies resulted at efficienciesof from 0.03% to 0.7%. Such colonies were selectively dissociated bytrypsinization when they reached approximately 1000 cells, using glasscloning cylinders and silicone hi-vac grease to affix the cylinders tothe petri plates. After trypsinization had liberated the cells from theplate, the cloned (colony purified) cells were removed from thecylinders with glass or plastic capillary pipettes together with a smallamount of the trypsin solution and plated in standard 60 mm diameterplasticware petri plates in CM diluted 1:1 with fresh Coon's 4506.07Medium as before. When these cells had grown to confluence, the wholeplate of cells was dissociated by trypsinization, diluted 1:6 or more,and transferred into fresh plates for subculture. The clonal cellstrains thus established were subsequently fed twice weekly with acomplete exchange of fresh 4506.07 growth medium without further needfor CM. Cloned cell strains cultured in this way were maintained for 25to 30 passages without signs of senescence or other failure of celldivision, and without any overt sign of transformation or geneticadaptation to continuous cultivation. Aliquots of these populations werefrozen for archival storage and the remaining cells were characterizedusing: (1) fluorescence immunocytochemistry (employing a double antibodytechnique, wherein the second antibody and purified hormone blockingcontrols were negative); and (2) RIA for insulin or C-peptide(indicators of beta cells), glucagon (indicator of alpha cells), andsomatostatin (indicator of delta cells).

Results from these assays indicated that some of the cells derived fromthe human pancreas that divide in Coon's 4506.07 medium may havepartially reverted to pluripotent cells that, in spite of clonalpurification, produce populations of cells that contain at least threeof the islet cell types: alpha, beta, and delta cells. In one assay, thecloned population showed clearly positive reactions (concentrated in theintracellular granules) indicating that the population was comprised of20-25% beta cells, 10-15% alpha cells, and 5% delta cells. The remainingcells were either negative or weakly positive for staining for thesethree cell types. In other cell strains, the result was that themajority of cells stained diffusely for each of these products. All ofthe cells in all of the clonal populations were strongly positive forthe neural and neuroendocrine marker, neuron specific enolase (NSE), andmost of the cells were strongly positive for a marker for secretorycells of neuroendocrine systems, chromogranin A. Some clones of humanpancreatic endocrine cells therefore can produce at least three of thecell types found in the normal adult, non-dividing islets of Langerhans.

In a similar experiment, clonal strains of human pancreatic islet cellsshowed specialization. Two clones of 27 tested, named HPSL-8U andHPSL-8D, apparently represented delta cells because when these clonedcultures were incubated for 24 hours in medium with no insulin and high(20 mM) glucose, they respectively produced 570 and 116 pg/ml ofsomatostatin (a distinctive hormonal product of delta cells). In highinsulin (15 μg/ml) and low glucose (2.5 mM) medium, these clonedcultures respectively produced only 9.6 and 28 pg/ml of somatostatin,thereby showing the anticipated lower response to these physiologicalconditions. Six of 27 clones produced low but significant amounts ofinsulin, ranging 88.5 to 114 pg/ml/24 hrs. None of the 27 clones madesufficient glucagon to be detected under these culture conditions.

EXAMPLE 6

This example illustrates the preparation of pancreatic endocrine cellstrain HPSL-6 and its steady-state production of insulin and C-peptideafter various population doublings.

Partially purified islets were prepared from pancreatic tissue collectedin St. Louis, using the method described in Example 2. Accordingly, theislets were concentrated by centrifugation, resuspended in Coon's4506.07 medium and distributed at densities of about 250 islets perstandard 100 mm plastic petri plate and fed with twice weekly changes ofCoon's 4506.07 medium. The cultures were maintained in water jacketedincubators set at about 36.5° C. and provided with a humidified, 5% CO₂in air gas mixture. Two weeks after initiating the cultures, the cellswere trypsinized, and the contents of one plate were distributed into 2new plates. The cells on these new plates were fed and incubated asbefore. The cells in these plates reached confluence (i.e., becamecrowded to the point of becoming contiguous, thereby ending log-phasegrowth of the culture) in 5 to 7 days, and again the cells from oneplate were trypsinized and passaged into 2 more plates (i.e., a 1:2passage ratio). In this way, the cells may be said to have undergone adoubling or one population doubling in each passage generation. Byconvention, population doublings (PDL) can be reckoned in thispassage-at-confluence method (as done here with HPSL-6) or by countingcells and diluting accordingly at each passage (as done with HPSL-8 inExample 7 below).

At each passage starting with PDL #13 (i.e., after 13 cell divisions ora 2¹³ -fold (approximately 8000-fold) expansion of the original cellpopulation) and continuing through PDL #18, the production of insulinand C-peptide were determined using standard radioimmunoassay procedures(using RIA kits from Peninsula Laboratories, Inc., Belmont, Calif.94002). The amount of insulin (striped bars) and C-peptide (blank bars)accumulated in the medium in 24 hours is presented in FIG. 1 (y-axis ishormone production per day, expressed as picograms hormone per 500 mlmedium per day; x-axis is population doubling values, expressed asP.D.L.). These are steady state values in the cultures and do notmeasure the hormone production in response to a glucose stimulus (seeExample 7 below). Apparently, after PDL #14 the amount of insulinproduced fell off sharply in these cultures, but did not disappearentirely. However, PDL #14 represents an approximately 16,000-foldexpansion of the cultured pancreatic endocrine cells, which is ample forthe production of thousands of individual grafts that can be derivedfrom a single donated pancreas.

EXAMPLE 7

This example illustrates the preparation of pancreatic endocrine cellstrain HPSL-8 and its steady-state production of insulin and C-peptideafter various population doublings.

Using the procedures outlined in Example 2 above, another set ofpartially purified islets, HPSL-8, were grown in serial culture in amanner similar to that set out in Example 6. This time, the initialinoculum was 300 to 500 islets per standard 100 mm diameter petri plate,and the passage ratios were 1:4 for each pass, which occurredapproximately every week. Thus, each passage corresponded to two PDL ora quadrupling of cell number. FIG. 2 shows bars that denote the amountsof insulin (stippled bars) and C-peptide (solid bars) produced by cellstrain HPSL-8 at various PDL states of the culture. The left y-axis,which is applicable to the depicted bars, has units of pg of hormone permg of cell protein per day (divided by a scale factor of 500) and thex-axis has units of days in culture. This bar graph is superimposed onthe cumulative growth curve (.sup.▪) for the first 73 days in culture ofthe cells. The right y-axis, which is applicable to the depicted curve,has units of cumulative number of cells in a logaritharic scale. Thevalues obtained at PDL #2-4 are unstimulated steady state values likethose shown for HPSL-6 in FIG. 1. The values obtained at PDL #8-10,#10-12, and #15-17 are calculated from the measured amount of insulinand C-peptide (determined by RIA) produced in a 15 minute periodfollowing a change to high glucose (20 mM) medium without added insulin.This measures the ability of cells in the culture to secrete insulinunder conditions of high glucose challenge, such as occurs in diabeticindividuals.

By PDL #8-10 (about 1000-fold expansion), there were no fibroblastsdetected in the expanded cultures, nor were there endothelial cells asjudged by the absence of immunofluorescent staining withanti-human-factor-VIII antibodies. Neuron-specific enolase (NSE), amarker for neuroendocrine cells, is seen in all cells of the cultures byimmunofluorescent staining. Similarly, another marker, also absent fromfibroblasts and endothelial cells, chromogranin A, was demonstrated inall cells of the culture after PDL #8-10. Prior to PDL #8-10, at PDL#2-4 (primaries), there were subsets of cells in the culture that didnot stain with these immunochemical reagents.

The population doubling time was about 2.7 days over the 73 days of thestudy. The amount of insulin produced in response to glucose challengewas found to be about 19 ng per mg cell protein per hour at PDL #8-10.It was also noted that the HPSL-8 monolayer cultures contain glucagonand somatostatin producing cells in addition to the insulin andC-peptide producing cells.

Hormone and C-peptide production in a series of 30 clones prepared fromHPSL-8 islets in passage 1 (PDL level 4-6) was assayed using the methodsdescribed in Example 5. Insulin was detectable in 6 clones (4 to 6 pg/mgcell protein/ml); no glucagon was found in any clone, and two clonesshowed high levels of somatostatin (160 and 500 pg/mg cell prot/hr).

EXAMPLE 8

This example illustrates that the HPSL-8 cells and islet or primaryculture cells display physiological similarity.

Time course assays using a standard RIA-type assay as in Examples 5, 6,and 7 were performed on culture medium of HPSL-8 cultures for insulin,C-peptide, glucagon, and somatostatin, and the results thereof are shownin FIGS. 3-5. At each time point in these graphs, four modifications ofthe basic Coon's 4506.07 medium formulation were used, whereby thetested cell culture was incubated in the modified medium for one weekprior to the glucose challenge described above. Modification A was lowcalcium (0.35 mM CaCl₂ •2H₂ O); modification B was low calcium plus 10μg/ml added human placental lactogen; modification C was high calcium(2.2 mM); and modification D was high calcium plus 10 μg/ml added humanplacental lactogen. The accumulation over time of insulin, C-peptide,glucagon, and somatostatin are illustrated in Graphs A, B, C, and D,respectively, of FIGS. 3-5. The y-axis is in units of hormoneaccumulated, namely pg hormone accumulated/mg cell protein/ml ± s.e.m.;the x-axis is in units of time, namely minutes.

With respect to the data depicted in FIG. 3, the hormones (or hormoneby-product in the case of C-peptide) were secreted into the medium bythe cultured HPSL-8 cells, and were measured following a 20 mM glucosechallenge in medium without added insulin. From FIG. 3A, it can be seenthat the accumulation of insulin secreted into the medium is paralleledafter a delay by C-peptide secretion, which indicates active processingof the prohormone to the active hormone. The same data expressed as arate of insulin secretion over time is presented in FIG. 4. From thisprofile, a pattern reminiscent of serum insulin values after glucosechallenge in an animal can be seen, a rise followed by an undershoot andreturn to an apparent basal level. The absolute timing is different, butthen the stimulation is also different in vitro from that of the in vivosituation because, at a minimum, there is no associated liver to act asa glucose/insulin repository in the culture vessel.

High insulin and low glucose (2.5 mM) stimulate the rate of glucagonsecretion, as seen in FIG. 5C. In FIG. 5B, which graphically displaysthe results of an experiment where the HPSL-8 cells were incubated inhigh insulin and low glucose, the production of C-peptide is shut off byhigh levels of insulin in the presence of low levels of glucose. Thenegative rates of insulin synthesis, shown in FIG. 5A, are interpretedas destruction and ligation and/or uptake of the initial high levels ofinsulin from the medium.

Somatostatin, which is seen at all stages of the cultures, showsvariations with differing levels of glucose; it seems to besignificantly increased at high insulin and low glucose levels, as shownin FIG. 5D.

In nearly every case, the highest hormone production was observed withthe modification B medium. It was particularly notable that high calciumhad a negative effect on the generation of insulin by the culturedcells. It was also notable that human placental lactogen, known toenhance insulin secretion by islets and isolated primary cells in vitro,had the same effect in vitro with the cultured cells. Therefore, theresponse of the cultured cells to the glucose challenge demonstrated thephysiological similarity of these monolayer cell cultures to isolatedislets even after an approximately 1000-fold expansion in vitro.

EXAMPLE 9

This example illustrates a method to assay the cytotoxicity of exogenousmaterials and bodily fluids that uses cultured pancreatic endocrinecells of the present invention. Cytotoxic agents generally, cytotoxicagents specific to pancreatic endocrine cells, and auto-antibodies inindividuals having no diabetic clinical symptoms can be assayed usingthe following procedures.

To measure the stimulatory effect on the basal release of insulin in thepresence of serum from diabetic patients or in the presence of someother test material, cells are cultured for 7 days in the presence ofeither 10% serum from normal individuals (control) or from testsubjects, at 8.3 mmol glucose. For non-serum test materials, cells arecultured for 7 days in the presence of either serial dilutions of thetest material or the diluent (control). Insulin release is measured inthe supernatant medium using standard RIA technology.

To measure the inhibitory effect on the high glucose induced, acuterelease of insulin in the presence of serum from symptomatic orpresymptomatic diabetes patients or in the presence of some other testmaterial, cells are cultured for 7 days in the presence of either 10%serum from normal individuals (control) or from test subjects. Fornon-serum test materials, cells are cultured for 7 days in the presenceof either serial dilutions of the test material or the diluent(control). At the seventh day, cells are challenged with 20 mmol or 5mmol glucose. Insulin release is measured in the supernatant at 15minute intervals after challenge to produce a time-course curve.

To measure antibody-dependent cytotoxicity, cultured pancreatic isletcells, prelabeled with sodium ⁵¹ Cr! chromate, are used as targets. Involumes of 50 μl, up to 5×10⁴ of the labeled target cells, are plated inquadruplicate in a 96 well assay plate. Effector cells (such as humanperipheral mononuclear cells) are added in ratios ranging from 100:1 to12.5:1 (effector:target cells), in the presence of purifiedimmunoglobulins from either normal donors (control) or test subjects.The plates are then incubated for 4 hours at 37° C. Supernatant fluid isharvested and counted in a gamma counter. Specific lysis may becalculated using the following formula: ##EQU1## where observed releaseis the mean radioactivity released in the presence of effector cells andsera, and spontaneous release is the mean radioactivity released fromtarget cells incubated in the medium alone. Total releasable activitymay be determined after treatment of the target cells with 2.5% TritonX-100.

To measure cellular-dependent cytotoxicity, cultured pancreatic isletcells, prelabeled with sodium ⁵¹ Cr! chromate, are used as targets. Involumes of 100 μl, up to 5×10⁴ of the labeled target cells, are platedin quadruplicate in a 96 well assay plate. Effector cells (such as humanperipheral mononuclear cells or sorted T cells from either normal donorsor test subjects) are added thereto in ratios ranging from 100:1 to12.5:1 (effector:target cells). MHC class I restricted activity isexcluded by testing the cells either with class I matched or nonmatchedcultures or in the presence and in the absence of anti-class I blockingantibodies. The plates are then incubated for 4 hours at 37° C.Supernatant fluid is harvested and counted in a gamma counter. Specificlysis may be calculated using the following formula: ##EQU2## whereobserved release is the mean radioactivity released in the presence ofeffector cells, autologous release is the mean radioactivity released bytarget cells incubated with 2×10⁵ unlabeled autologous cells in place ofeffector cells, and total releasable activity is the total amount ofradioactivity incorporated in target cells.

EXAMPLE 10

This example illustrates a method for altering blood sugar levels in amammal in need of altering its blood sugar levels that uses culturedpancreatic endocrine cells of the present invention.

Late passage cultivated islet cells of the present invention as coherentaggregates of cells (pseudoislets) or suspended cells were incubated inanimal collagenous matrix that was caused to gel, thereby formingpseudotissues suitable for grafting into a host animal. In particular,HPSL-8 cells of PDL #19-21 were suspended in an isotonic neutralcollagen solution which was allowed to gel at 37° C. for three hours,thereby forming cell-type pseudotissues composed of about 6.5×10⁶ totalcells each. HPSL-8 cells of PDL #23 also were reaggregated spontaneouslyby gentle rotation of suspended cells in an Erlenmeyer flask at 37° C.for three days. During these three days, the cells reaggregated intogroups of from about 20 to about 250 cells, forming pseudoislets oftightly adherent islet-like spherical masses. These masses were furtherembedded in a collagen gel as above, resulting in pseudoislet-typepseudotissues.

Severe combined immune deficiency (SCID) mice that were homozygous atthe SCID locus and whose blood sugar was assayed over a period of up to58 days were used to demonstrate that the above-described pseudotissueswork in vivo to restore normoglycemia, as shown in FIG. 6 (wherein they-axis unit is mg % ± s.e.m. for measurement of glycemia, and the x-axisunit is days). Blood sugar determinations were made (approximately)twice weekly using the Ames Glucostix and the Ames Glucometer II on adrop of blood from a cut in the tail vein of each mouse. First, thesubject mice were caused to be diabetic by administering to each mouse afreshly dissolved solution of streptozoticin ("STZ" in FIG. 6), anestablished procedure for experimentally causing a mouse to be diabeticby preferentially killing the insulin-producing pancreatic beta cells.The animals were observed for about two weeks to ensure that their bloodsugar levels rose to the diabetic range, which is taken to be greaterthan 300 mg per 100 ml. The mice were then supplied with a subcutaneousgraft of human pancreatic culture cells of the present invention (HPSL-8at PDL #19-21) in the region of the dorsal fat pad (between the shoulderblades).

The result with host mouse 24S (FIG. 6A), using a graft of culturedhuman islet cells bound into a cell-type pseudotissue, is a clearexample of a successful graft of greatly expanded human islet cells. Theblood sugar became regulated down to normal levels very rapidly andremained there for at least three weeks, the duration of the experiment.Host mouse 20S, which received a graft of a pseudoislet-typepseudotissue, appears to have received a successful graft as well,although not as profoundly so (see FIG. 6B). This mouse required anextra administration of the streptozotocin to induce its diabeticcondition. Nevertheless, over the course of the experiment, it is clearthat blood sugar levels were regulated after the implant of the graft ofthe pseudotissue.

Post mortem examination of the pancreas of host mouse 24S showed thatvery few beta cells survived, as anticipated. This examination occurred56 days after the streptozotocin treatments. Standard histologicaltechniques were used, namely immunohistochemical staining for insulin ofhistological sections of the host mouse's pancreas. In the graft(located beneath the skin by a marker of blue tatoo ink included at thetime of grafting), it was possible to see human cells that were heavilystained for insulin and were clustered around capillaries that hadinvaded the grafted tissue. It is known that human beta cells applythemselves directly to islet capillaries, whereas in mouse and pigislets, the beta cells are usually one or two cell layers removed fromthe capillaries. Human cells were unequivocally identified by indirectimmunofluorescence using monoclonal antisera directed against humanclass I histocompatibility antigens. These observations demonstrateclearly that the human cells of the graft were able to establishabundant insulin synthesis and storage and to organize themselvescharacteristically with respect to the capillaries even after anapproximately 1,000,000-fold expansion in cell culture. The human cellsof the graft apparently were instrumental in restoring the normoglycemiaobserved in the mouse immediately after grafting.

EXAMPLE 11

This example illustrates methods to culture and clone normal humanthyroid cells according to the present invention.

Coon's 4506.035 or Coon's 4506.07 medium was prepared as in Example 1,except that the concentration of MgCl₂ was adjusted to 0.48 mM, theconcentration of hydrocortisone was adjusted to 0.01 mM, theconcentration of selenous acid was adjusted to 2 ng/ml, thetriiodo-thyronine concentration was adjusted to 3 pg/ml, bovinehypothalamus extract was added to a final concentration of 75 μg/ml, andbovine pituitary extract was added to a final concentration of 5 μg/ml.

All preparation and treatment of thyroid tissue was performed understerile conditions, similar to the procedures used for rat cellsreported in Ambesi-Impiombato et al., Proc. Natl. Acad. Sci. USA,77:3455-3459 (1980). Normal human thyroid tissue, obtained from an organdonor, was freed of thyroid tissue attached thereto from adherentconnective tissue, cut into small (less than 1 mm diameter) pieces,washed in Ca⁺⁺ - and Mg⁺⁺ -free Hanks' balanced salt solution (HBSS) bya 5 minute centrifugation at 500× g, and dissociated enzymatically. Theenzymatic digestion was performed according to the method of Coon, Proc.Natl. Acad. Sci. USA, 55:66-73 (1966), for which a solution is preparedconsisting of 20 U/ml collagenase, CLSPA (Worthington, Freehold, N.J.),0.75 mg/ml trypsin, 1:300 and 2% heat-inactivated dialyzed chicken serum(Gibco) in Ca⁺⁺ - and Mg⁺⁺ -free HBSS (hereinafter referred to as "CTCsolution"). The digestion was done in a shaking water bath at 37° C. fortwo hours, after which the tissue was mostly a cell suspension. Largerfragments were allowed to settle for 2 minutes at 1× g. Supernatantswere collected, and then cells and small fragments of follicles wereseeded at a density of 10⁵ cells per 100 mm plastic tissue culture dish(Falcon, Becton Dickinson, Lincoln Park, N.J.).

Secondary cultures were made by incubating monolayers in CTC solutionfor about 25 minutes at 37° C., after washing in Ca⁺⁺ - and Mg⁺⁺ -freeHBSS. For cloning, single cell suspensions were plated at 10² -10⁴ cellsper 100 mm dish. Cloning plates were fed with medium conditioned bypreincubating 12 ml of fresh medium for 24 hours in crowded plates ofthe "parental" mass cell populations. Individual, well-isolatedepithelial colonies arisen from previously marked single cells weretrypsinized selectively using cloning cylinders.

These culture procedures and media yielded proliferating thyroid cellcultures from different human donors. Neither the presence of 6% FCS,the pituitary extract alone, nor the hypothalamus extract alone weresufficient to sustain the growth of human thyroid cells. In the presenceof serum, without any extract or with either one of them, cells wereunable to divide (at least not appreciably), and the cytoplasm becameswollen and very pale. Each culture showed noticeable differences in therequirements for pituitary extract, as compared to pancreatic isletcells. In most instances, 50 μg/ml of pituitary extract was evidently inexcess. As observed under phase-contrast microscopy, cells becamegradually larger, contained evident stress-fibers, and ultimately died.Pituitary extracts added to a concentration of 5 μg/ml or less supportedhealthy-appearing cultures.

EXAMPLE 12

This example illustrates assays used to characterize the thyroid cellcultures of the present invention, and provides results of such assaysand general observations that relate to the HNTB-2K clonal cell strain.

Thyroglobulin (Tg) production was determined in the supernatants by astandard immunoradiometric assay method using a commercial kit (Henning,Berlin, Germany) according to manufacturer's instructions.

For chromosomal counts, 2 hours after medium changing, cells weretreated with 10 μg/ml demecolcine (Colcemid, Calbiochem, La Jolla,Calif.) for 3 hours, released by enzymatic treatment with CTC solutionas disclosed in Example 11, centrifuged, and resuspended in hypotonicsolution (4 parts of 5.6 gr/l KCl and 1 part of 7.3 gr/l CaCl₂ •2H₂ O).After 15 minutes, cells were fixed by progressively adding 0.1 ml, 0.2ml and 0.5 ml of fixative (methanol/acetic acid, 3:1, vol/vol) to thecell suspension, followed by 5 minutes centrifugation at 1000 ×g andpouring off or aspirating the supernatant. Up to 5 ml fresh fixative wasthen added, dropwise under gentle shaking, followed by centrifugationand elimination of the supernatant, as above. Three more fixing cycleswere performed by repeating the above procedure. Fixed cells were thenspread on microscope slides, and 25 metaphase-staged cells were observedusing phase contrast optics and a drawing attachment.

The ability of TSH, alone or in the presence of insulin, to stimulatecell growth was tested by 3H-thymidine incorporation. TSH-induced3H-thymidine incorporation was assayed as described in FRTL5Today--Proceedings Of The First International Workshop OnCharacterization And Standardization Of An In Vitro Thyroid Cell System,(Ambesi-Impiombato and Perrild, eds., Elsevier Science Publishers, 1989)(hereinafter "FRTL5 Today") with minor modifications, as follows: Normalhuman thyroid cells (HNTB-2K) and cultured rat thyroid cells (FRTL5)were seeded in 24 multiwell plates at densities of 5×10⁴ and 4×10⁵cells/well, respectively, in complete medium. After 24 hours, the cellswere washed three times in Ca⁺⁺ - and Mg⁺⁺ -free HBSS and then shiftedto 0.5% FCS, extract-free medium, with no added TSH. After 7-14 days,cells were washed twice in Ca⁺⁺ - and Mg⁺⁺ -free HBSS and incubated 72hours at 37° C. in 0.5 ml/well of medium with no thymidine, containing0.1% bovine serum albumin (BSA) (Janssen, Olen, Belgium), 2.5 μCi/ml3H-thymidine (Amersham, Arlington Heights, Ill.), no insulin or 4 μg/mlinsulin, no TSH or bovine TSH (Sigma), at concentrations varying from10⁻⁷ M to 10⁻¹³ M. At the end of incubation, cells were washed twice inCa⁺⁺ - and Mg⁺⁺ -free HBSS and twice with 0.5 ml/well of ice-cold 10%trichloracetic acid. After removal of supernatants, 0.5 ml/well of 2%sodium dodecyl sulfate was added, and 10 minutes later supernatants wereanalyzed for incorporated 3H-thymidine by liquid scintillationspectroscopy.

TSH-induced cAMP accumulation was assayed as described in FRTL5 Todaywith minor modifications, as follows: HNTB-2K and FRTL5 cells wereseeded in complete medium at densities of 5×10⁴ and 2×10⁵ cells/well,respectively, in 96 multiwell plates. After incubation for 24 hours, thecells were washed three times in Ca²⁺ - and Mg²⁺ -free HBSS and thenshifted to 0.5% FCS extract-free medium, with no added TSH. After 7-14days, cells were washed twice in Krebs-Ringer buffer and incubated 1hour at 37° C. in 0.1 ml/well of the same buffer, with 0.1% BSA(Janssen), 2 mg/ml glucose, 0.5 mM 3-isobutyl-1-methylxanthine, andbovine TSH (Sigma) at concentrations varying from 10⁻⁷ to 10⁻¹³ M. Thereaction was stopped by removing the incubation medium and adding 0.1ml/well of 70% ethanol. After 20 minutes at room temperature, plateswere centrifuged, supernatants were transferred to plastic tubes, andthe ethanol was evaporated at 40° C. The quantity of cAMP was determinedby a commercial radioimmunoassay kit (Diagnostic Products Corporation,Los Angeles, Calif.) according to manufacturer's instructions.

The following chart lists thyroglobulin production by different clonesof thyroid cells derived from normal human donors:

    ______________________________________                                        Cell Line      PDL    TG (ng/cell/day)                                        ______________________________________                                        HNTB-1         20     1317                                                    HNTB-1 CL A    20     18                                                      HNTB-1 CL D    20     25                                                      HNTB-1 CL F    20     21                                                      HNTB-1 CL G    18     22                                                      HNTB-1 CL G    20     28                                                      HNTB-1 CL K    15     92                                                      HNTB-1 CL J    20     19                                                      HNTB-2         18     1267                                                    HNTB-2 CL A    15     28                                                      HNTB-2 CL B    15     46                                                      HNTB-2 CL C    15     77                                                      HNTB-2 CL E    15     18                                                      HNTB-2 CL F    15     44                                                      HNTB-2 CL H    15     18                                                      HNTB-2 CL I    15     20                                                      HNTB-2 CL K    15     1234                                                    HBTB-2-CL K    20     1262                                                    HNTB-2 CL J    15     22                                                      ______________________________________                                    

The clone morphology of HNTB-2K was not homogeneous when observed inphase-contrast microscopy, and was influenced by the proliferative stateof the cells: non-confluent, log-phase cultures showed mostly elongated,rather pale cells, while at confluence they became more like classicalepithelia, showing darker cytoplasm and many secretory granules inside.The karyotype showed a normal diploid number of chromosomes in allmetaphases counted. In the complete medium, the population doubling timeof the cells of the HNTB-2K clone was 58 hours.

At all concentrations tested, TSH, alone or in the presence of insulin,was unable to stimulate acutely ³ H-thymidine incorporation, in contrastto the rat system (FRTL5) where TSH is reportedly a mitogenic factor.FIG. 7 shows the acutely stimulated ³ H-thymidine incorporation by FRTL5(control; cross-hatched bars) and the lack of response by HNTB-2K(stippled bars) cells in the presence of TSH at various concentrations,alone and with insulin at a concentration of 5 μg/ml. Values areexpressed as tissues over control.

Acutely added TSH was able to induce a dose-dependent, up to 10-foldincrease, of cAMP accumulation in HNTB-2K cells. The stimulation wasevident, within 1 hour, even at very low concentrations (10⁻¹¹ M). Thebehavior of HNTB-2K cells in this assay is remarkably similar to that ofrat thyroid cells in the same assay. FIG. 8 shows the TSH-stimulateddose-dependent increase of cAMP accumulation in FRTL5 (control; closedcircles) or HNTB-2K (open circles) cells. Cells that received variousconcentrations of TSH were assayed for concentration of cAMP per mgtotal protein, as indicated on the y-axis of FIG. 8.

The HNTB-2K cells appear not to be transformed because they do notexhibit any of the usual symptoms of transformed cells: (1) they do notgrow in soft agar; (2) they have retained a diploid karyotype; (3) theyhave shown no decrease in serum or extract requirement for growth; (4)as part of another experiment HNTB-2K did not make tumors in 45 daysafter two SCID mice received grafts of 5×10⁶ and 10⁷ cells; and (5) thecloning efficiency has not increased with successive generations inculture.

EXAMPLE 13

This example illustrates the culturing of parotid cells in accordancewith the present invention.

Culture medium was prepared according to Example 1. Two organ donatedsamples (≦2 gm) of normal parotid gland and 2 surgical samples fromnormal appearing portions of the parotid gland were removed when thewhole gland was resected for cancer therapy. In all four samples of theapparently healthy parotid gland from different adult humans, parotidcells were cultured successfully. The tissue samples were put intoculture within 3 hours after removal from the patients.

In each case, the method of transferring the cells to culture wassubstantially the same. One to two grams of healthy tissue weredissected and minced by repeated snips of blunt tipped "iris" scissorsat the edge of a tilted petri dish. When the tissue was reduced to 1-2mm³ bits, a trypsin-collagenase digestion mixture was added (2-4 ml),and the tissue was incubated for 1-4 hours at 37° C. and also for 12-15hours (overnight) at room temperature (21° C.). During this digestionthe tissue was reduced to single cells and small pieces of the glandulartissue consisting of 50-100 cells. After vigorous mixing to break up theclusters further, the cells were washed in fresh medium (Coon's4506.07). Cells and fragments were plated in from 3-10 100 mm plastictissue culture petri plates and cultured in 12 ml of Coon's 4506.07medium for 7-10 days in humidified, 5% CO₂ atmosphere incubator at 36.5°C.

An alternative method for the very small amounts of tissue that arederived from a needle biopsy has been used, whereby the salivary glandcells and minced pieces (about 1 mm³) were embedded in a gel made fromreconstituted basement membrane according to Kleinman et al.,Biochemistry, 25:312 (1986). Reconstituted basement membranes arecomposed of an extract of EHS mouse chondrosarcoma tumors, which consistlargely of type IV collagen. This extract of extracellular material,with a biochemical composition similar to that of normal basementmembrane or lamella, was shown by Kleinman et al. supra, to promote thegrowth and differentiation of a wide variety of epithelial cells, and isavailable commercially from Collaborative Biomedical, a division ofBecton Dickinson, Lincoln Park, N.J., as "Matrigel™".

In either primary culture situation, the salivary cells grow out inabout a week or two, at which time they are treated with trypsin andcollagenase, washed, and diluted into new, secondary cultures(designated passage 1 or P1). After the cells have spread out on thesurface of the plate or under and within the meshes of the reconstitutedbasement membrane gel, they may be released with minimal cell damage,using trypsin and collagenase and then inoculated into fresh plates atsplit ratios of from 2-10 to 1 or diluted and plated at 500, 1000, 2500,5000 cells/100 mm plate for cloning. Plating efficiencies varied withthe donor from a low value of 0.01% to a high value of 0.1%. After twoto three weeks of culture in homologously conditioned medium (CM),colonies were isolated, grown into populations (clonal cell strains)that were routinely fed twice weekly with a complete exchange of freshmedium 4506.07. Aliquots of these populations were frozen for archivalstorage and the remaining cells were assayed for salivary glycoproteins:gustin and lumicarmine using indirect immunocytochemistry (all sampleswere clearly positive while a negative control, human normal thyroidcells, was negative). Enzyme assays showed that the cells which weretested at passage 6 (P6) secreted 1820 IU/ml amylase activity into themedium. Amylase is a characteristic marker for parotid gland secretion.On the basis of the findings of these three major salivary proteins, thecultures were found to be well differentiated human parotid gland cellsthat would be suitable for grafting.

The contents of each of the references cited in the present application,including publications, patents, and patent applications, are hereinincorporated by reference in their entirety.

The present invention being thus described, it will be obvious that thesame may be varied in many ways. Such variations are not to be regardedas a departure from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

What is claimed is:
 1. A method of altering blood sugar levels of amammal comprising administering expanded human pancreatic cells to themammal, which expanded cells have been expanded in a cell culture andcomprise enriched, non-transformed, serially passaged human pancreaticendocrine cells, wherein said cell culture is substantially free offibroblast, macrophage, and capillary endothelial cells, and whereinsaid cell culture is prepared by a method comprising the steps of:(a)selecting said pancreatic endocrine cells from a human tissue thatcomprises said cells; (b) concentrating said selected cells; (c)resuspending said concentrated cells in a culture medium, wherein saidmedium comprises a basal medium supplemented with (i) hypothalamusextract and (ii) pituitary extract; (d) culturing said resuspendedcells; and (e) passaging said cultured cells periodically to expand saidculture.
 2. The method of claim 1, wherein said cell culture is a clonalcell culture of pancreatic endocrine cells prepared by a methodcomprising the steps of:(a) culturing the cell culture obtainedaccording to the method of claim 1; (b) growing said culture; (c)dissociating said cells; (d) inoculating said disassociated cells into aculture vessel that contains medium conditioned by pancreatic endocrinecells for a first plating and culturing said inoculated cells to producecolonies of cells; (e) harvesting individual colonies of cells; (f)inoculating said colonies into a culture vessel for a second plating andculturing said inoculated cells; and (g) serially passaging the cells ofstep (f).
 3. The method of claim 1, wherein said cells arematrix-embedded.
 4. The method of claim 3, wherein said cells areembedded in a gel matrix.
 5. The method of claim 1, wherein said mammalis human.
 6. A method of altering blood sugar levels of a humancomprising administering expanded pancreatic cells of human origin tothe human, which expanded cells have been expanded in a cell culture andcomprise enriched, non-transformed, serially passaged pancreaticendocrine cells, wherein said cell culture is substantially free offibroblast, macrophage, and capillary endothelial cells, and whereinsaid cell culture is prepared by a method comprising the steps of:(a)selecting pancreatic endocrine cells from a human tissue that comprisessaid cells; (b) concentrating said selected cells; (c) resuspending saidconcentrated cells in a culture medium, wherein said medium comprises abasal medium supplemented with (i) hypothalamus extract, (ii) pituitaryextract and (iii) one or both of human placental extract or humanplacental lactogen; (d) culturing said resuspended cells; and (e)passaging said cultured cells periodically to expand said culture.
 7. Amethod of altering the blood sugar of a mammal comprising administeringto said mammal non-transformed mammalian cells obtained from anon-transformed cell culture, wherein said cell culture comprises:agrowth medium comprising (i) a basal medium supplemented with (ii)hypothalamus extract, (iii) pituitary extract, and (iv) placentalextract and/or a placental lactogen, and an enriched, expanded,non-transformed population of human pancreatic endocrine cells.
 8. Themethod of claim 7, wherein said pancreatic endocrine cells do not growin soft agar, and do not form tumors in 45 days after about 10⁷ cellsare implanted into SCID mice.
 9. The method of claim 8, wherein saidpancreatic endocrine cells are obtained from an adult.
 10. The method ofclaim 9, wherein said culture is substantially free of fibroblasts,macrophages, and capillary endothelial cells.
 11. The method of claim10, wherein said pancreatic endocrine cells secrete insulin at a steadystate and at a greater rate in response to glucose stimulation.
 12. Themethod of claim 11, wherein said pancreatic endocrine cells are matrixembedded.
 13. The method of claim 12, wherein said pancreatic endocrinecells are aggregated to form pseudoislets.
 14. The method of claim 13,wherein said pseudoislets comprise pluripotent cells.